ripa binding buffer  (Thermo Fisher)

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPR/Cas9 induces DNA damage and apoptosis in PGCs. (A) Flow cytometry analysis 24 h after electroporation, quantifying the proportion of Annexin V + /PI + cells. The horizontal axis indicates PI and the vertical axis Annexin V. The upper-left quadrant (Annexin V + /PI + ) represents late apoptotic cells, and the lower-right quadrant (Annexin V + only) represents early apoptotic cells. Upper panels: results after electroporation with Cas9 + various gRNAs; lower panels: results with dCas9 + various gRNAs. (B) Bar graph of Annexin V + /PI + percentages across groups. Cas9 editing induced a highly significant increase in late apoptosis. (C) γ-H 2 AX foci (green) detected by immunofluorescence 24 h after electroporation. Foci appear as discrete nuclear puncta; nuclei are counterstained with DAPI (blue). Scale bar = 10 µm. (D) Quantification of γ-H 2 AX foci per cell. Cas9 targeting resulted in a significant increase in γ-H 2 AX foci per cell, whereas dCas9 with sgRNA did not. Statistical significance determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: After another centrifugation, the supernatant was removed and cells were resuspended in 500 μL of 1× Annexin V Binding Buffer (from Annexin V-FITC/PI Apoptosis Kit, Elabscience E-CK-A211).

Techniques: CRISPR, Flow Cytometry, Electroporation, Immunofluorescence

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: PGCs are more sensitive to DNA double-strand breaks than somatic cells. (A) Cell viability curves after 24 h treatment with increasing concentrations of etoposide (ETP; x-axis shows log 10 [ETP]). PGC viability dropped sharply even at low ETP doses, whereas CEF cells were more tolerant (viability data are mean ± SD of triplicates). (B) Apoptosis detection in PGCs treated with low-dose ETP. The proportion of Annexin V + /PI + cells (late apoptosis) was significantly elevated even at 0.03 µM ETP. (C) Western blot analysis of γ-H 2 AX protein in PGCs treated with varying ETP concentrations (left panel; β-actin as loading control). The bar graph (right) shows the ratio of γ-H 2 AX to β-actin band intensity, with a marked increase at 3 µM ETP (p-values are indicated in the figure). (D) Quantification of γ-H 2 AX foci per cell in THP-1 cells, male PGCs, and female PGCs after exposure to X-ray doses of 0, 2, 4, 6, and 8 Gy. After 48 h recovery, γ-H 2 AX foci increased significantly with higher radiation in THP-1, male PGCs, and female PGCs. Female PGC data points are red squares; male PGCs are blue squares; THP-1 are black circles. (E) Cell-cycle distribution of male vs. female PGCs after DNA damage. PGCs were irradiated (2, 4, 6 Gy), cultured 48 h, and analyzed by flow cytometry for cell-cycle phase (propidium iodide staining). Stacked bars show the percentage of cells in G 0 G 1 , S, and G 2 /M phases in untreated vs. irradiated cells. After damage, female PGCs predominantly arrested in G 2 /M (increased G 2 fraction), whereas male PGCs accumulated in S phase. Statistical significance by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: After another centrifugation, the supernatant was removed and cells were resuspended in 500 μL of 1× Annexin V Binding Buffer (from Annexin V-FITC/PI Apoptosis Kit, Elabscience E-CK-A211).

Techniques: Western Blot, Control, Irradiation, Cell Culture, Flow Cytometry, Staining

Journal: Cellular and Molecular Immunology

Article Title: Oncolytic virus M1 reinvigorates CD8 + T-cell immunity against glioblastoma through B-cell-dependent antigen cross-presentation in the spleen

doi: 10.1038/s41423-026-01396-w

Figure Lengend Snippet: OVM exhibits antiglioma activity. A Glioma cell lines GL261, CT2A, U-87 MG, and U-118 MG were infected with OVM at MOIs of 0.001, 0.01, 0.1, 1, and 10, respectively. Cell viability was assessed via the MTT assay at 24, 48, 72, and 96 h postinfection. B Glioma apoptosis was evaluated by Annexin V/PI staining following infection with OVM at an MOI of 1 for 48 h. C Quantification of early apoptotic cells (Annexin V + PI − ) and late apoptotic cells (Annexin V + PI + ). D Schematic of the in vivo treatment schedule. Glioma cells (GL261, CT2A, or GL261-Luc) were intracranially implanted on Day 0, followed by daily tail vein injections of vehicle or OVM from Day 5 to Day 9. E Bioluminescence imaging of GL261-Luc tumors was performed via the IVIS Spectrum system on days 13, 17, and 20. F Tumor burden was quantified by total flux (photons/second). OS of GL261 ( G ) and CT2A ( H ) orthotopic tumor-bearing mice treated with OVM was assessed and represented by Kaplan‒Meier survival curves. I Kaplan‒Meier survival curves of GBM patients from the CGGA database stratified by MXRA8 expression. J Analysis of MXRA8 expression across classical, mesenchymal and proneural GBM subtypes in the CGGA database. K Human glioma tissue fragments (~1 mm³) were treated with OVM (150 µL, 1.16 × 10 8 CCID 50 /mL), vehicle, or TMZ (50 mg/mL) for 72 h. Viral replication was quantified via qPCR, and replication coefficients were calculated as the ratio of viral copies in standard culture to those in parallel inactivated controls; a coefficient >2 indicated productive viral replication. Tissue viability was assessed by MTT staining

Article Snippet: The cells were washed once with precooled PBS, recentrifuged, and resuspended in 100 μL of 1× Annexin V Binding Buffer (from the Annexin V-APC/PI Apoptosis Kit, Elabscience, E-CK-A217) to a concentration of 1 × 10 6 cells/mL.

Techniques: Activity Assay, Infection, MTT Assay, Staining, In Vivo, Imaging, Expressing